Determining glycated proteins is very important in diagnosing and controlling diabetes. In particular, recent studies on hemoglobin A1c have proved that risk of occurrence and progression of complications is significantly lowered by controlling the level to 7% or less, so that the level is often used as an index that is essential in clinical fields. As a quantification method for hemoglobin A1c, there are generally known electrophoresis, ion-exchange chromatography, affinity chromatography, immunization, and enzymatic methods. However, the electrophoresis and chromatography methods require expensive dedicated devices, and processing speeds thereof are low, so that those methods are inappropriate for clinical examinations to process many samples. Meanwhile, the analysis method of the immunization method is relatively easy and can be performed in a small amount of time, so that the method has rapidly spread in recent years. However, the method is performed using an antigen-antibody reaction, so that it is problematic that the accuracy is not always good due to reproducibility and effects of coexisting substances.
Meanwhile, the enzymatic method has been suggested as a determination method that requires no dedicated device, has a high processing speed, and is highly accurate, easy, and inexpensive (JP-A-08-336386, WO 97/13872, JP-A-2001-95598, JP-A-2000-300294, and Clinical Chemistry 49(2): 269-274 (2003)).
Hemoglobin is glycated at an ε-amino group of intramolecular lysine and at α-amino groups of valine in α- and β-chain N-terminals, but hemoglobin A1c is hemoglobin where an α-amino group of valine in a hemoglobin β-chain N-terminal has been glycated (definition accepted as the international standard in Clinical Chemistry and Laboratory Medicine 40(1): 78-89 (2002)). Therefore, in order to specifically determine a glycated β-chain N-terminal of glycated hemoglobin without a separation operation using a protease and a ketoamine oxidase, it is believed that specificity is required in enzymatic reactions of either or both of a protease and a ketoamine oxidase.
Specifically, glycated hemoglobin has three glycated sites, that is, intramolecular lysine, α-chain N-terminal, and β-chain N-terminal, so that in order to determine only the glycated β-chain N-terminal, it is necessary that those enzymes be combined according to specificity as below.
That is, in the case where proteases and ketoamine oxidases are classified into (P1) to (P4) and (K1) to (K5), respectively, it is necessary that those enzymes be combined as below: <(P1) and (K1) or (K2) or (K3) or (K4)>, <(P2) and (K1) or (K3)>, <(P3) and (K1) or (K2)>, <(P4) and (K1)>, <(P3) and (K5) and (K3)>, and <(P3) and (K5) and (K4)>.
The properties of the respective classified enzymes are as follows.
(P1) cleaves a glycated amino acid and/or a glycated peptide only from a glycated β-chain N-terminal of glycated hemoglobin, (P2) cleaves a glycated amino acid and/or a glycated peptide only from glycated α- and β-chain N-terminals of glycated hemoglobin, (P3) cleaves a glycated amino acid and/or a glycated peptide only from a glycated β-chain N-terminal of glycated hemoglobin and a site including a intramolecular lysine, (P4) cleaves a glycated amino acid and/or a glycated peptide from a glycated α- and β-chain N-terminals of glycated hemoglobin and a site including a intramolecular lysine, (K1) reacts only with a glycated amino acid and/or a glycated peptide derived from a glycated β-chain N-terminal of glycated amino acids and/or glycated peptides cleaved from glycated hemoglobin by a protease to be used in combination, (K2) reacts only with a glycated amino acid and/or a glycated peptide derived from glycated α- and β-chain N-terminals of glycated amino acids and/or glycated peptides cleaved from glycated hemoglobin by a protease to be used in combination, (K3) reacts only with a glycated amino acid and/or a glycated peptide derived from a glycated β-chain N-terminal and from a site including intramolecular lysine of glycated amino acids and/or glycated peptides cleaved from glycated hemoglobin by a protease to be used in combination, (K4) reacts with a glycated amino acid and/or a glycated peptide derived from glycated α- and β-chain N-terminals and from a site including intramolecular lysine that have been cleaved from glycated hemoglobin by a protease to be used in combination, and (K5) reacts with a glycated amino acid and/or a glycated peptide from a site including intramolecular lysine without reacting with a glycated amino acid and/or a glycated peptide derived from a glycated β-chain N-terminal of glycated amino acids and/or glycated peptides cleaved from glycated hemoglobin by a protease to be used in combination.
However, as proteases for generating a glycated amino acid and/or a glycated peptide, which serves as a substrate for a ketoamine oxidase, from glycated hemoglobin or a fragment thereof, there have already been known proteases described in JP-A-08-336386, WO 97/13872, JP-A-2001-95598, JP-A-2001-57897, WO 00/50579, WO 00/61732, Clinical Chemistry 49(2): 269-274 (2003), etc. However, there is no description about specificity to cleave a glycated amino acid and/or a glycated peptide from a glycated β-chain N-terminal without substantially cleaving a glycated amino acid or a glycated peptide from a glycated α-chain N-terminal from glycated hemoglobin or a fragment thereof.
Meanwhile, an angiotensin-converting enzyme described in JP-A-2000-300294 is also estimated to react with a β-chain N-terminal glycated tripeptide from its known substrate specificity, but there is no specific description that shows cleavage of a glycated amino acid and/or a glycated peptide from a glycated β-chain N-terminal without substantially cleaving a glycated amino acid and/or a glycated peptide from a glycated α-chain N-terminal of glycated hemoglobin or a fragment thereof, for example, there is no description that shows results of quantification of specificity of the angiotensin-converting enzyme to an α-chain N-terminal glycated tripeptide or to a β-chain N-terminal glycated tripeptide.
Meanwhile, in JP-A-2000-300294, there is no description showing that trypsin, proline-specific endoprotease, and carboxypeptidase P, which were used in generating a β-chain N-terminal glycated tripeptide from glycated hemoglobin, generate no glycated amino acid or no glycated peptide derived from a site including intramolecular lysine and a glycated α-chain N-terminal.
Furthermore, the inventors of the present invention have confirmed that an angiotensin-converting enzyme hardly cleaves fructosyl valine from a β-chain N-terminal glycated tripeptide under a general reaction condition, so that the angiotensin-converting enzyme is not considered to be a protease that cleaves a glycated amino acid and/or a glycated peptide from a glycated β-chain N-terminal without substantially cleaving a glycated amino acid or a glycated peptide from an α-chain N-terminal.
As described above, there has not been known a protease and a reaction condition for a protease that cleave a glycated amino acid and/or a glycated peptide from a glycated β-chain N-terminal without substantially cleaving a glycated amino acid or a glycated peptide from a glycated α-chain N-terminal of glycated hemoglobin or a fragment thereof, that is, a protease that has the above-described specificity (P1) or (P3) or a reaction condition for a protease that is designed so as to accomplish the above-described specificity (P1) or (P3).
Meanwhile, in general, the following screening method is viewed as a method of screening a protease that cleaves a glycated amino acid and/or a glycated peptide from a glycated β-chain N-terminal without substantially cleaving a glycated amino acid or a glycated peptide from a glycated α-chain N-terminal of glycated hemoglobin or a fragment thereof, that is, a screening method for a protease that has the above-described specificity (P1) or (P3). That is, glycated hemoglobins are divided into hemoglobin where an α-chain N-terminal has been glycated and hemoglobin where a β-chain N-terminal has been glycated, and a protease that selectively cleaves a glycated amino acid and/or a glycated peptide only from the hemoglobin where a β-chain N-terminal has been glycated when using those hemoglobins as substrates is searched using an enzyme (such as a ketoamine oxidase) that reacts with the glycated amino acid and/or the glycated peptide cleaved by the protease, based on coloring. However, the glycation rate of a glycated hemoglobin product existing in nature is low (about 5%). Therefore, the yield in separating hemoglobin where an α-chain N-terminal has been glycated and hemoglobin where a β-chain N-terminal has been glycated was extremely low, and it was difficult to detect the activity of the protease using those substances as substrates because hemoglobin is red. As described above, an easy and effective screening method has never been known.
On the other hand, ketoamine oxidases include the following enzymes.
1) A ketoamine oxidase that is derived from a microorganism belonging to the genus Fusarium (JP-A-07-289253), the genus Gibberella, the genus Candida (JP-A-06-46846), or the genus Aspergillus (WO 97/20039) and mainly reacts with ε-1-deoxyfructosyl-L-lysine (hereinafter also referred to as FK) or a peptide including it and fructosyl valine (hereinafter also referred to as FV),
2) a ketoamine oxidase that is derived from a microorganism belonging to the genus Corynebacterium (JP-A-61-280297), the genus Penicillium (JP-A-08-336386), or the genus Trichosporon (JP-A-2000-245454) and mainly reacts with FV. In general, a step for cleaving FV from a hemoglobin β-chain N-terminal glycated peptide using a protease has disadvantages in that the reaction hardly proceeds in general and must be performed using a large amount of enzymes for a long time, so that, in order to overcome such disadvantages, a ketoamine oxidase that reacts also with a glycated peptide cleaved from a hemoglobin β-chain N-terminal glycated peptide by a protease has been required as a ketoamine oxidase to be used in determining hemoglobin A1c. Thus,
3) a mutant ketoamine oxidase derived from Corynebacterium JP-A-2001-95598) and a ketoamine oxidase that is derived from a microorganism belonging to the genus Achaetomiella, the genus Achaetomium, the genus Thielabia the genus Chaetomium, the genus Gelasinospora, the genus Microascus, the genus Coniochaeta, or the genus Eupenicillium (EP 1,291,416) and reacts with 1-deoxyfructosyl-L-valyl-L-histidine (hereinafter also referred to as FVH), which have the above-described property, have been reported in recent years.
However, a ketoamine oxidase belonging to 1) has the property (K4) and a ketoamine oxidase belonging to 2) has the property (K2), but there is no description that they react with FVH. A ketoamine oxidase belonging to 3), even a ketoamine oxidase that is derived from a microorganism belonging to the genus Eupenicillium and reacts with FK at the most low rate, reacts with FK at a rate of 9.78% in the case where the reaction with FVH is defined as 100% (EP 1,291,416). Therefore, it is considered that the ketoamine oxidase sufficiently reacts with a glycated amino acid and/or a glycated peptide from a site including intramolecular lysine cleaved from glycated hemoglobin by a protease, and there is no description about a reaction with a glycated peptide derived from a glycated α-chain N-terminal cleaved from glycated hemoglobin by a protease, for example, 1-deoxyfructosyl-L-valyl-L-leucine (hereinafter also referred to as FVL), so that it is not considered to be a ketoamine oxidase having the property (K1), (K2), or (K3).
As described above, there have not been reported ketoamine oxidases that: have the property (K1); have the property (K2) and reacts with FVH; or have the property (K3).
Meanwhile, in order to prepare ketoamine oxidases that have the property (K1), and have the property (K2) and reacts with FVH, it is considered to perform modification of known ketoamine oxidases by amino acid substitution, deletion, insertion, etc. However, it has not been known which amino acid residue in the primary structure of the enzyme contributes to reduction of a reaction with FK or FZK. Therefore, the activity to FK or ε-1-deoxyfructosyl-(α-benzyloxycarbonyl-L-lysine) (hereinafter also referred to as FZK) cannot be reduced by modification of any ketoamine oxidase gene, i.e., there has not been known preparation of ketoamine oxidases that have the property (K1), and have the property (K2) and reacts with FVH by modification.
Meanwhile, there has not been known reduction of a rate of the activity to FK or FZK compared to that to FVH by regulating a reaction condition for a ketoamine oxidase capable of reacting with FVH.
In order to clearly distinguish and determine glycation of an α-amino group of valine in a β-chain N-terminal existing in glycated hemoglobin using a protease and a ketoamine oxidase, the specificity of the protease and ketoamine oxidase must be combined as described above. However, in JP-A-08-336386, WO 97/13872, JP-A-2001-95598, and Clinical Chemistry 49(2): 269-274 (2003), there is no description about specific determination of a glycated β-chain N-terminal of hemoglobin, and there is only a description that the value that was obtained or may be obtained by the HPLC method significantly correlates with the determined value obtained by the disclosed enzymatic method. Moreover, there is no mention about specificity of the used protease and ketoamine oxidase, and a glycated amino acid and/or a glycated peptide cleaved simply by degrading glycated hemoglobin by a protease is detected by a ketoamine oxidase, so that it is considered that there was detected a mixture of hemoglobin where an ε-amino group of intramolecular lysine and α-amino groups of valine in α- and β-chain N-terminals have been glycated. Furthermore, in examples in JP-A-2001-95598, glycated hemoglobin was determined using a protease and a ketoamine oxidase that reacts with FVH. However, centrifugation was performed as an operation, and there is no description that the determination can be performed without a separation operation.
JP-A-2000-300294 suggests an enzymatic method of specifically determining only hemoglobin in which an α-amino group of valine in hemoglobin β-chain N-terminals has been glycated. In this method, sequential processing was performed by a protease capable of cleaving the carboxyl group side of leucine at the third position from a hemoglobin β-chain N-terminal and then by a protease capable of cleaving His-Leu from fructosyl-Val-His-Leu, to thereby generate fructosyl valine, and the amount of glycation of an α-amino groups of valine in a hemoglobin β-chain N-terminal was specifically determined. However, this method have the following disadvantages: it requires two stages of protease reactions; it is difficult to strictly control the protease reaction for cleaving the carboxyl group side of leucine at the third position from a hemoglobin β-chain N-terminal in the first stage; and a reaction of the step for cleaving an α-glycated amino acid from a hemoglobin β-chain N-terminal glycated peptide in the second stage hardly proceeds in general and must be performed using a large amount of enzymes for a long time. Moreover, in the method shown in examples, a cumbersome separation operation (ultrafiltration) was performed twice, and there is no description that the method of the present application enables specific determination of a glycated β-chain N-terminal of glycated hemoglobin without a separation operation.
EP 1,291,416 suggests a method of determining a glycated protein such as hemoglobin A1c with an oxidase capable of reacting with FVH to be released by a protease such as Molsin, AO-protease, Peptidase (available from Kikkoman Corporation), carboxypeptidase Y, or Protin P (available from Daiwa Kasei K.K.). The description further suggests, in the case where FK generated by a protease is problematic, determination by an oxidase capable of reacting with FVH after elimination of FK by a fructosyl amine oxidase that reacts with FK, or determination using an oxidase that reacts FVH and hardly reacts with FK. However, there is no mention about distinction between a glycated amino acid and/or a glycated peptide derived from a glycated α-chain N-terminal of glycated hemoglobin, and there are not demonstrated examples on determination of a glycated β-chain N-terminal of glycated hemoglobin by the suggested method. In addition, there is no description that the suggested method can be performed without a separation operation.
Although the above-described determination methods relating to glycated hemoglobin were known, there have not been known a method and a reagent kit for specifically determining a glycated β-chain N-terminal of glycated hemoglobin using enzymes without a separation operation.